Molecular Cloning of Mouse OB Gene and Its High Expression in E.coli

In this study,mouse OB cDNA has been amplified by RT-PCR technique.The cDNA of mouse OB gene was subcloned into pTO-T7 plasmid and the recombinant plasmid has been transformed into Escherichia coli BL21(de3) and induced by IPTG,the induced product was identified by western blotting and purified by affinity chromatography technique.The results showed that the mouse leptin fusion protein with a his-tag was about 20 ku.When the IPTG concentration was 0.5 mmol/L,the expression products accounted for more than 50% of the total E.coli protein after 2.5 hours inducing in 37℃.After purification,the purity of the mouse leptin fusion protein could accounted for more than 90%.Western blot analysis indicated that the fusion protein could react specifically with anti-leptin body.